It has been known that 2-keto-L-gulonic acid can be produced biosynthetically from D-sorbitol or L-sorbose. For example, in Japanese patent Publication No. 40,154/1976, it is reported that 2-keto-L-gulonic acid can be produced from D-sorbitol with the aid of microorganisms belonging to the genus Acetobacter, Bacterium or Pseudomonas which are capable of oxidizing the hydroxymethyl radical at the 1-position and the hydroxymethylene radical at the 2-position of D-sorbitol to convert those radicals into a carboxyl radical and a carbonyl radical, respectively, under aerobic conditions. However, according to the said process, the yield of 2-keto-L-gulonic acid is less than 6 g/l. Moreover, in "Acta Microbiologica Sinica" 21 (2) 185-191 (1981), it is reported that 2-keto-L-gulonic acid can be accumulated in a mixed culture broth of two microorganisms, i.e. Pseudomonas striata and Gluconobacter oxydans at the concentration of 30 and 37 g/l starting from 70 g/l and 100 g/l, respectively, of L-sorbose. However, it is reported that Gluconobacter oxydans alone produced trace amount of 2-keto-L-gulonic acid and Pseudomonas striata produced none at all.